Average Dissociation Constant Research Articles

Lymphocytotoxic antibodies in systemic lupus erythematosus: studies of their temperature dependence, binding characteristics, and specificity in vitro.

The lymphocytotoxicity of 33 lupus sera was tested against purified helper/inducer (OKT4) and cytotoxic/suppressor (OKT8) subsets of T lymphocytes at 15 degrees C and 37 degrees C in vitro. There was significantly less killing of both OKT4 and OKT8 cells at 37 degrees C (p less than 0.001 and p less than 0.01) and the ratio of OKT4/OKT8 cell killing at 15 degrees C (1.39 (0.73); mean (SD] was different from that observed at 37 degrees C (0.79 (0.42)) (p less than 0.001). OKT4 killing was greater than OKT8 killing in 21 out of 33 sera at 15 degrees C, while 22 of these sera showed predominantly OKT8 cytotoxicity at 37 degrees C. The relation between the OKT4/OKT8 cell ratio and OKT4/OKT8 serum killing was examined in 22 patients at both temperatures: a significant inverse correlation was observed at 37 degrees C (r = -0.53; p = 0.015) but not at 15 degrees C (p greater than 0.05). The addition of metabolic and cytoskeletal inhibitors increased cytotoxicity at 37 degrees C, but not IgM surface binding. A Scatchard binding analysis of the reaction at 15 degrees C showed that large numbers of antibody molecules were bound to both subsets, with a low average dissociation constant of less than or equal to 6 x 10(-8) mol/l, and electrophoretic blotting indicated that the target surface antigens varied in type and number among individual lymphocytotoxic sera. The demonstration of temperature dependent, tight binding between lymphocytotoxic antibody and variable antigens on the T cell surface emphasises the potential for this phenomenon to affect lymphocyte function in vivo in patients with systemic lupus erythematosus.

Stoichiometry of lac repressor binding to nonspecific DNA: three different complexes form.

The stoichiometry of lac repressor binding to nonspecific DNA was investigated by three different techniques. Four molecules of the fluorescent probe 5,5'-bis(8-anilino-1-naphthalenesulfonate) [bis(ANS)] bind to each repressor subunit with an average dissociation constant of 20 microM. Nonspecific DNA displaces most of this bound bis(ANS), reducing the fluorescence. Titrations of repressor with nonspecific DNA monitored with high [bis(ANS)] (5-15 microM) had end points at 8 base pairs per repressor. Lower [bis(ANS)] (0.1-1 microM) resulted in end points at either 15 or 26 base pairs per repressor, depending on the ionic strength. These end points correspond to complexes containing approximately one, two, or four repressors per 28 base pairs. Boundary sedimentation velocity experiments with saturating amounts of repressor revealed that five repressors can bind to 28 base pairs. By monitoring the circular dichroism as DNA was added to repressor, the sequential appearance of complexes containing approximately four, two, and one repressors per 28 base pairs was observed. The inability of repressor cores or iodinated repressor to bind to complexes containing one or two repressors per 28 base pairs implies that all of the repressors directly contact the DNA in the complex containing four repressors per 28 base pairs. It is proposed that while two subunits of each repressor contact the DNA in complexes containing one or two repressors per 28 base pairs, only one subunit of each repressor contacts the DNA in the complex with four repressors per 28 base pairs. These results suggest a novel mechanism for the one-dimensional diffusion of repressor along DNA.

Quantification of Progesterone Binding in Mammary Tissue of Pregnant Ewes

Progestin-binding sites in mammary tissue from 14 prepartum, multiparous ewes at 50, 80, 115, and 140 d of gestation were demonstrated by the binding of [3H] R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) to ovine mammary cytosol in the presence of sodium molybdate and excess cortisol. Homogenization extracted 89% of total mammary receptors (nuclear) into cytosol. Binding was specific for progestins and was of high affinity. The average dissociation constant for [3H]R5020 specifically bound to receptors extracted into mammary cytosol was 1.9 (± .4) × 10−9M (n=14) and did not change significantly over the test period. However, binding capacities (fmol/mg cytosolic protein) differed according to stage of gestation with averages of 125±53, 149±26, 656±216, 57±22 at 50, 80, 115, and 140 d of pregnancy, respectively. Increased number of progestin-binding sites at 115 d of gestation (whether data are expressed per unit of tissue weight, DNA, or cytosolic protein) suggests that an increase per mammary epithelial cell may be necessary to produce the full lobuloalveolar proliferation observed at this stage of gestation.

Activation of memory and virgin B cell clones in hyperimmune animals.

To study the long-term memory response, BALB/c mice were allowed to rest for over a year after a secondary immunization with the hapten 2-phenyl-oxazol-5-one (phOx). For the tertiary immunization two different protocols were used. In one protocol mice were injected i.v. and 3 days later spleen cells were fused to a nonproducing hybridoma line. PhOx-specific hybridomas were established and the sequence of the heavy and light chain mRNA was determined. This tertiary response resembled the diversity pattern of the secondary response with a further increase both in somatic mutations and in the average dissociation constant. The high number of somatic mutations demonstrates the persistence of memory B cell clones over a long time period. In the second protocol mice were boosted with an i.p. injection of alumprecipitated antigen phOx and 7 or 14 days later spleen cells were fused. Sequence analysis of heavy and light chain mRNA showed that these tertiary response antibody molecules had surprisingly few somatic mutations, indicating an activation of virgin B cell clones in these hyperimmunized animals. The maturation of these newly stimulated B cell clones seems to follow somewhat similar rules to those found for the primary response. It appears therefore that the two immunization protocols reflect the response of memory and virgin B cells, respectively.

Anti-dihydropyridine Antibodies Exhibit [3H]Nitrendipine Binding Properties Similar to the Membrane Receptor for the 1,4-Dihydropyridine Ca2+ Channel Antagonists

The [H]nitrendipine binding properties of antidihydropyridine antibodies were characterized and compared with the known [H]nitrendipine binding properties of the membrane receptor for the 1,4-dihydropyridine Ca channel antagonists. Immunization of rabbits with dihydropyridine-protein conjugates resulted in the production of antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca channel antagonists. Anti-dihydropyridine antibodies were found specifically to bind [H]nitrendipine, [H]nimodipine, [H]nisoldipine, and [H]PN200-110 with high affinity, while [H]verapamil, [H]diltiazein, and [H]trifluoperazine were not recognized. [H]Nitrendipine binding to antidihydropyridine antibodies was characterized by a competitive radioimmunoassay. The average dissociation constant of the [H]nitrendipine-antibody complex was 0.06 ± 0.02 nM for an antiserum studied in detail, and ranged from 0.01 to 0.24 nM for all antisera studied. The average dissociation constant determined from rate constants of association (k1 = 2.7 × 10 M s) and dissociation (k = 1.8 × l0s) of [H]nitrendipinefronianti-dihydropyridine antibodies immobilized on Protein-A Sepharose was 0.66 nM Inhibition of [H]nitrendipine binding was specific for the 1,4-dihydropyridine Ca channel modifiers, and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM; structurally unrelated Ca channel antagonists did not modify [H]nitrendipine binding to the anti-dihydropyridine antibodies. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca channel antagonists and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca channel antagonists.

Calcium fluxes and calcium buffering in human neutrophils.

Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.

High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers.

Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind [3H]nitrendipine, [3H]-nimodipine, [3H]nisoldipine, and [3H]PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while [3H]verapamil, [3H]diltiazem, and [3H]trifluoperazine were not recognized. The average dissociation constant of the [3H]nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of [3H]nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify [3H]nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace [3H]nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

Prostaglandin E and F2α receptors in human myometrium during the menstrual cycle and in pregnancy and labor

The binding of prostaglandins E1 and F2α has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2α binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2α, respectively. Scatchard analysis of tritium-labeled prostaglandin F2α binding was also linear, but the affinity to these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2α binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites. In addition to confirming the presence of specific prostaglandin E and F2α binding sites in the myometrium, these data also suggest: (1) that specific prostaglandin E binding sites are present in the cervix and (2) that the concentrations and affinities of prostaglandin E binding sites do not change during the menstrual cycle and are similar before and during labor in pregnant patients at term.

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains.

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes.

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.

Androgen receptors are similar in fetal and adult rabbits.

In an effort to explain the separate roles of testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) in virilizing the male fetus, we compared the binding of these androgens to cytosolic receptors from urogenital tract tissues of fetal and adult male rabbits. As measured by a direct binding assay, fetal and adult androgen receptors are similar in respect to specificity, affinity, and amount of binding. Apparent dissociation constants for dihydrotestosterone binding averaged 1.1 nM for fetal receptor and 0.8 nM for adult androgen receptors. Average apparent dissociation constants for testosterone binding were 4- to 24-fold higher than those for dihydrotestosterone in fetal and adult tissues. Nonradioactive dihydrotestosterone and testosterone competed for [3H]dihydrotestosterone binding to the androgen receptor in both adult prostate and fetal urogenital sinus in a manner consistent with their affinity for binding, whereas estradiol, progesterone, and cortisol were weak competitors for [3H]dihydrotestosterone. On sucrose density gradients, both testosterone and dihydrotestosterone were bound to a protein with a sedimentation coefficient of approximately 8S. Although androgen receptors were detectable in urogenital tubercle and urogenital sinus of both male and female fetuses on days 18 and 29 of gestation, we were unable to characterize androgen binding in fetal Wolffian ducts. The nature of the androgen receptor in this tissue remains unresolved. These findings are consistent with the hypothesis that dihydrotestosterone formation acts to amplify the androgenic signal in both the fetus and adult, but is not absolutely required for virilization.

Suspension culture of human mast cells/basophils from umbilical cord blood mononuclear cells.

Selective growth of human mast cells/basophils was obtained in suspension cultures of mononuclear cells from umbilical cord blood. A fraction of culture supernatant of phytohemagglutinin-stimulated T cells, which lacked interleukin 2, was required for the selective growth of mast cells. When the mononuclear cells were cultured for 2-4 wk in the presence of the fraction, 50-90% of the total cells in the cultures contained metachromatic granules. Under the optimal culture conditions, the number of mast cells/basophils recovered from the cultures was 30-60% of the number of mononuclear cells plated. Cultured mast cells/basophils bear 1.2-3.83 X 10(5) IgE receptors per cell and contained 0.48-1.6 micrograms of histamine per 10(6) cells. The average forward rate constant, k1, and dissociation constant, k-1, for the binding of human IgE to IgE receptors on the cells were 1.9 X 10(5) M-1 sec-1 and 6.9 X 10(-5) sec-1, respectively (average equilibrium constant = 2.75 X 10(9) M-1). Specific binding of human IgE with high affinity indicates that the cells recovered in the suspension culture are human mast cells/basophils. Cultured cells sensitized with human IgE released a substantial amount of histamine upon exposure to anti-IgE. The results indicate that human mast cells/basophils obtained in the culture are functionally mature.

Polarographic study on interaction between human IgG and sheep antihuman IgG antiserum and its analytical application.

The Brdiccka current (Polarographic catalytic hydrogen evolution current produced by proteins in the presence of cobalt salt) of human immunogloblin G (IgG) and sheep antihuman IgG antiserum (anti-IgG) was studied by the d.c. and differential pulse Polarographic techniques. The Brdicka current for a mixture of IgG and anti-IgG was smaller than the sum of the currents for IgG and anti-IgG. This difference was attributed to the complex formation between IgG and anti-IgG. Anti-IgG could be titrated with IgG (or vice versa) by Polarographic techniques based on the Brdicka current. The average intrinsic dissociation constant of the IgG–anti-IgG complexes was estimated from the titration curve. The Polarographic method based on the Brdicka current and coupled with immunoreaction is useful to determine trace amounts of antigen (or antibody) down to 10−10 m using the differential pulse Polarographic technique.

Effect of Metal Ions on the Structure and Activity of Calcium-Activated Neutral Protease (CANP)1

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.

Inhibition of the plasma membrane [H+]-ATPase of Neurospora crassa by N-ethylmaleimide. Protection by nucleotides.

Pre-incubation of the plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent, N-ethylmaleimide (NEM), leads to a marked inhibition of ATPase activity. Loss of activity depends upon preincubation pH and follows pseudo-first order kinetics with respect to NEM concentration. MgATP, the physiological substrate for ATPase activity, protects against NEM inactivation with an average dissociation constant of 1.5 mM at 0 degrees C. This value agrees well with the measured Km for MgATP hydrolysis (1.3 mM at 30 degrees C and 0.9 mM at 15 degrees C). MgADP also protects against NEM inhibition with an average KD of 0.18 mM at 0 degrees C; MgADP is a competitive inhibitor of enzyme activity, with a Ki of 0.08 mM at 30 degrees C and 0.09 mM at 15 degrees C. Free ATP and ADP, as well as other Mg nucleotides (MgGTP, MgCTP, and MgUTP) which are hydrolyzed at much slower rates than MgATP, exert smaller protective effects. These results suggest that nucleotides protect against NEM inhibition by binding to the catalytic site of the ATPase. NEM can therefore be used as a probe to study the nature of enzyme-ligand interactions.

Prostaglandin E 2 receptor in the myometrium: Distribution in subcellular fractions

[ 3H]Prostaglandin (PG) E 2 bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE 2 and PGE 1 competed for the [ 3H]PGE 2 binding site. The PGs inhibited in the following decreasing order: PGE 2 = PGE 1 ⪢ PGF 2α > PGA 2 > PGF 1β > PGB 2. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10 −9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10 −11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10 −11 mol binding sites/g. The results suggest that there is a high-affinity PGE 2 receptor present in both plasma membrane and sarcoplasmic reticulum.

Thyroglobulin structure-function: Anilinonaphthalene sulfonate binding and the definition of nonpolar sites on native bovine thyroglobulin

Summary Native bovine thyroglobulin has been shown by fluorescence titration and equilibrium dialysis to bind 6 moles of 1,8 anilinonaphthalene sulfonate (ANS) per mole of protein at pH 7.0 in 0.1 M sodium phosphate buffer. The average dissociation constant for this binding is 3 × 10 −5 M. Analysis of the binding data indicates significant cooperativity in the interaction with the reaction order, J, near 2. The binding of ANS to bovine thyroglobulin is accompanied by a blue shift in the ANS fluorescence maximum of near 50 nm and an increase in fluorescence yield of approximately 60 fold.

The detection of β-adrenoceptors on murine lymphocytes

The β-adrenoceptor antagonist ( 125I)-iodohydroxybenzyl pindolol was used to detect and characterise β-adrenoceptors on the surface of mouse lymphocytes by a direct radioligand receptor assay using intact viable cells. The binding was stereoselective and appeared to be with a β 2-type receptor as shown by competition with a number of adrenergic reagents. Scatchard analysis of the ligand binding demonstrated approximately 3000 binding sites per cell with an average dissociation constant ( K d ) of 0.92 nM. Little variation in binding characteristics was observed between lymphocytes from individual mice of either sex or in 24 different inbred mouse strains. In addition no significant differences were noted between T and B enriched lymphocyte populations, i.e. there was no functional polymorphism of the β-adrenoceptor on mouse lymphocytes. Thymocytes carried a lower affinity receptor which may be related to their functional immaturity. By using antisera specific for murine cell surface alloantigens, the β-adrenoceptor was shown not to be associated with the H-2K, H-2D, Ia, Ly−1, −2, −3, −4, or −5, Tla or Thy antigens. However, specific antisera for the Ly-7 antigen did partially inhibit IHYP binding. The results show that lymphocytes have a β 2-adrenoceptor which closely resembles the receptor on other tissues.

Receptors for glucocorticoids in the lens epithelium of the calf.

The calf lens epithelium contains a specific cytoplasmic receptor for glucocorticoids. This binding protein has a high affinity for dexamethasone (average dissociation constant, 8 x 10(-9) mole per liter), a low capacity (average, 550 femtomoles per milligram of protein), extreme heat sensitivity, and exhibits a pattern of competition similar to that of glucocorticoid receptors in other tissues. This provides direct biochemical evidence that these tissues may function as a target organ for glucocorticoids.

Interaction of human Clq with insoluble immunoglobulin aggregates

A system of interaction between 125I-Clq and IgG aggregates insolubilized by glutaraldehyde, which was designed as a model for studying the initial event in activation of the complement sequence, is described. Binding of Clq was shown to be rapid, specific, reversible and saturable, with an average dissociation constant in the order of 1 × 10 −8 M. The binding data produced non-linear Scatchard plots. The binding was favored at an ionic strength below μ = 0.15. a pH range of 5.5–8.5. and was sensitive to low concentrations of organic solvents. Among many compounds which interfered with Clq-IgG aggregate interaction, an anionic compound, suramin, was shown to be a good inhibitor of Clq binding with a sigmoidal inhibition curve and with a K D of Clq-suramin complex in the order of 5 × 10 −5 M. Dissociation of radiolabeled Clq by succinylation caused a loss of binding activity but the succinylated Clq retained the capacity to inhibit 125I-Clq binding to IgG aggregates. The characteristic radioiodination pattern of Clq subunits was shown to be significantly changed when iodination was carried out on the IgG aggregates-bound Clq in such a fashion as to imply an IgG aggregates-induced conformational change in Clq, suggesting the usefulness of this system in exploring the possible mechanism underlying immune-complex mediated Cl activation.